DE2164626C3 - 08/31/71 Japan 66282-71 Microbiological process for the simultaneous production of a straight-chain dicarboxylic acid, an omega-hydroxy fatty acid and an (omega-1) -keto fatty acid - Google Patents

Description

Are acrylic acid, its salts or anion exchange resins or mixtures of acrylic acid and their salts with an anion exchange resin, “As is preferred, the culture medium in the process added according to the invention, the amount of straight-chain dicarboxylic acids, omega-hydroxy fatty acids and (omega-l) -ketocarboxylic acids increased about 5 times compared to that if none of these additives are used.

The normal paraffins with at least 10 carbon atoms, which are used in the culture medium, cores used either individually or in mixtures will. The preferred η-paraffins have 10 to 20 carbon atoms and include, for example Nensäure- or tartaric acid form, can be used.

The microorganism strains used in the process according to the invention can be n-paraffin exploited as the only source of carbon. The strain Corynebacterium dioxydans nov. sp. (MC-1-l strain) was also sold under FERM-P No. 690 from Fermentation Research Institute, Ministry of Trade and Industry, Japan, and the strain Corynebacterium hydrocarbooxydans nov. sp. (JA-1 strain), which is a gram positive Stab bacterium, under FERM-P No. 800 deposited at the same institute.

These two strains of microorganisms have been registered with the American Type Culture Collection under ATCC 21766 (internal name MC-1-l strain) and ATCC

n-Decan.n-Dodeca ^ n-Tridecan.n-Tetradecan.n-Pen- 15 21767 (internal name JA-1-Stamm) deposited.

tadecane, n-hexadecane, n-octadecane and n-eicosane.

The amount of η-paraffins used is usually 10 to 100 g, preferably 15 to 80 g, and in particular 20 to 70 g per liter of culture medium.

Any nitrogen source that can be used to grow micro-ao Organisms is suitable can be used in the method according to the invention as nitrogen source. Examples of nitrogen sources are: alkali nitrates, such as sodium nitrate or potassium nitrate; Ammonium salts of inorganic acids, such as ammonium nitrate, Amonium sulfate, ammonium chloride, or ammonium phosphate; Ammonium salts of organic acids, such as ammonium acetate, ammonium lactate or ammonium tartrate; Urea; Ammonia; Yeast extracts; Meat extracts and corn steep liquor. These compounds can be used alone or in the form of mixtures can be used as nitrogen source. The amount of the nitrogen source used is usually between about 0.1 and 10 g and preferably between 0.5 and 8 g per liter of culture medium.

Examples of mineral salts which can be used in the method according to the invention are NaH ₂ PO ₁ , Na ₂ HPO ₄ , Na ₃ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , K ₃ PO ₄ , CaHPO ₄ and others Phosphates. They are used either individually or in mixtures. It is also possible to use magnesium, iron, manganese, calcium, copper, zinc, molybdenum and boron salts of inorganic acids which provide ions of these metals. They are also used either individually or in mixtures. Specific examples are: MgSO ₄ -7 H ₂ O, FeSO ₄ · 7 H ₂ O, CaCl ₂ , CaCO ₃ , Ca (OH) ₂ . MnSO ₄ · 5 H ₂ O, CuSO ₄ · 5 H ₂ O, CuCl ₂ , ZnSO ₄ · 7 H ₂ O, MoO ₃ , H ₃ BO ₃ and sea water. The amount of mineral salts depends on the type used and is usually between about 5 / <g and 10 g per liter of culture medium.

The yield of the products can in the process according to the invention by incorporation at least an additive from the group of acrylic acid, whose salts, such as sodium, potassium, ammonium or calcium salt, and anion exchange resins, preferably by incorporating at least Acrylic acid or one of its salts and at least one anion exchange resin, increased in the culture medium will.

Any commercially available anion exchange resin e.g. B. a quaternary ammonium salt, a primary Amine, a secondary amine, a tertiary amine or a polyamine as an exchange group and the Performing anion exchange reaction can be used. The anion exchange resin can z. B. in the hydroxyl form, the chloride form, the weak acid form, such as phosphoric acid, Zitro-Die microbiological properties of these microorganisms are given below.

Corynebacterium dioxydans nov. sp. Strain MC-1-1 (ATCC 21766)

1. Rods, 0.6 to 1.2 by 3.0 to 9.5 microns. Cell branching and coke-like shapes are determined.

No spore formation. Not able to move by itself. Gram positive.

2. Nutrient agar colony: circular, smooth, undivided, convex raised, pale pink to orange.

3. Nutrient agar slope: moderate growth, filiform, shiny, reddish-pink.

4. Nutrient broth: fragile membrane, cloudy, excess sediment.

5. Nutrient gelatin stick: no liquefaction.

6. Litmus milk: alkaline, not peptonized.

7. B. C. P. milk: alkaline, not peptonized.

8. No indole formation.

9. No formation of nitrite from nitrate.

10. Hydrogen sulfide formation.

11. Starch is not hydrolyzed.

12. Methyl red test: negative.

13. No acetylmethylcarbinol formation.

14. Catalase: positive.

15. No ammonia formation.

16. Citrate is used as the only source of carbon.

17. Aerobic growth.

18. Optimal temperature 30 to 35 ⁰ C.

19. Growth at pH 5.0 to 9.0.

20. Acid formation from glycerine, glucose, fructose and mannitol.

Neither acid nor gas formation from xylose, lactose, sucrose, starch, maltose, galactose, mannose, Arabinose and cellulose.

The above microbiological properties were determined according to the classification standards, described in Bergey's Manual of Determinative Bacteriology, 7th Edition, examined and it was found that the above strain belongs to the genus Corynebacterium. The morphological and Physiological properties of the above strain were matched with the descriptions in Bergeys

5 6

Manual, but no identical could be found. The microbiological properties of the above-

Strain that would be described therein can be found. named Stanuns were according to Bergey's Manual of The above strain differs from Determinative Bacteriology, 7th edition examined, Corynebacterium glutamicum ncv. sp., Corynebac and it was found that the above strain teriuin liümn, sov. sp., Corynebacterium petrophilum 5 (JA-I) belongs to the genus Corynebacterium. the iiov. sp. and Corynebacteriuin acetoglutamicum nov. morphological and physiological properties sp. in size, different breeding properties of the above-mentioned strain were treading, compared to the effect on milk and the formation of turbulence in Bergey's handbook, hydrogen sulfide could be used or the fermentation of carbohydrates. but no identical tribe described in it Heel / differs from the above trunk also io would be found.

of Corynebacterium hydrocarboclastus nov. sp. in this tribe differs from Corynebacte-

the size, the breeding properties, the ability to produce rium petrophilum nov. sp. to reduce Corynebacterium aceto-nitrate, and the ability to carbohydrates glutamicum nov. sp., Corynebacterium lilium nov. sp. to ferment. Furthermore, and Corynebacterium acetoacidophilum nov. sp. in the above-mentioned stem of Corynebacterium, the fermentation of carbohydrates and the formation of rantiacum nov. sp. and Corynebacterium roseura nov. Hydrogen sulfide. It also differs from sp. in size, breeding characteristics, the Corynebacterium hydrocarboclastus nov. sp., Coryne's ability to reduce nitrate, drr ability to Schwe- bacterium paraaldehydium nov. sp. and to form corynebacte hydrogen, or the ability to produce carbonium flavo-aurantiacum nov. sp. to ferment in the fermentation of hydrates. Consequently, the ao carbohydrates, the effect on milk, the Reduk-MC-1-l strain (FERM P No. 690; ATCC 21766) as a cation of nitrate, etc., and of Corynebacterium also became a new species of the genus Corynebacterium. rantiacum nov. sp., Corynebacterium acetophilum j. This became Corynebacterium dinov. sp. and Corynebacterium roseum nov. sp. in the

ι oxydans nov. sp. called. Fermentation of carbohydrates, the decomposition of

35 starch or the reduction of nitrate. Consequently became

! ru. · U * u λ the above-mentioned strain JA-I (FERMP. No. 800;

C ^ rynebactenumhydrocarbooxydansnov.sp. _A TCC 21767) as belonging to a new species of the genus

Mamrn ja-i ^ aica, uibt) regarded as belonging to Corynebacterium. This was

1. Rods, 0.7 to 1.2 by 4.0 to 8.0 microns. Corynebacterium hydrocarbooxydans nov. sp. ge cells branching and kokkenähnliche forms ^{3o na "nt} · ... · be ti Found No sporulation Not ^Dcr P ^H -... ^{of the} medium is usually at 3 out of itself motile Gram positive adjusted ^{to 9,} preferably 5.. to 8.5, and in particular to 6 to 8. The growing temperature is

2. Nutrient agar colonies: circular, smooth, unsupported usually 20 to 40 ⁰ C and preferably 20 to divided to undulating, convex raised, orange 35 37 c. If it is particularly desired, the omegabis reddish-orange. Obtaining hydroxy fatty acid in large quantities will

3. Nutrient agar slope: moderate growth, a temperature between about 23 and 30 C, shiny, reddish pink. admitted- The cultivation time is usually around

/ 1 vi-u ♦ au -uu uv u χα t. ti 2 days. If desired, the cultivation can be more than

FÄ ?? ς. ^zerbr f ^hllche membrane clear _{+0 8 days, such as 10} days to 2 weeks, durchuberschussiges sediment. _{filled di} £ _rt ^ _{n jst djes jed} * _{ch mcht necessary Ge.}

5. Nutrient gelatinous: no liquefaction. homely can be with a breeding time of about 2 to

6. Litmus milk: unchanged. ^{10 days cn} - ^{the goal} - ^d · ^h - ^{the formation} S ^and accumula

tion of these 3 acids in the culture medium in full

7. BCP-Müch: unchanged. ₄₅ catch can be achieved.

. 8. No indole formation. Breeding can be done in any known way

ο v »;" » u-iA, «, "mV. wt. be performed. A culture medium similar to the above

9. No formation of nitrite from N. occurred. specified carbon source, nitrogen source and

10. Hydrogen sulfide formation. Contains mineral salts, is adjusted to the desired pH

11. Starch is not hydrolyzed. ⁵ ° ^value set and then by compressing

th water vapor at a temperature of example

12. Methyl red test: negative. _{around 120} ° c in a period of for example

13. No acetylmethylcarbinol formation. sterilized for 10 to 30 minutes. The sterilization

14 Catalase · insitive ^can, for example, also be carried out in this way

ι *. Naiaiase. positive. _{g5 that you can for several days at 100} ° C for

15. No ammonia formation. sterilized intermittently for 1 hour each. Subsequent

16 is citrate as a sole carbon source comparable ^ηά ^ ^{one of the} aforementioned strains ^is inoculated into the culture medium and _evaluates at the above temperature under aerobic conditions (examples

17. Aerobic growth. _{6o, for} example, as a culture of shaking or as a culture that

18. Optimal temperature: 30 to 35 ° C. is mixed by air supply).

ίο \ i7 "" u * u ·· ",",, _Λ . . " _N As stated above, the common

19. Growth be. e.nem pH 6.0 to 9.0. Use of at least one of the compounds

20. Acid formation from glycerol, glucose, lactose, from the group of acrylic acid and its salts, and Sucrose, maltose, fructose and mannose. 65 at least one anion exchange resin in the culture neither Acid or gas formation from xylose, medium preferred. The addition of these compounds

■ Starch, galactose, mannitol, arabinose and cellulic in every stage prior to cessation of the dissolute. take place. It is possible to use these additives too

sterilize and add them to the culture medium before or after 2 to 3 times higher in the event that an anion expression

one of the above strains or exchange resin is added to the inoculation and more than about 5 times

to be added during breeding. The preferred higher in the case that acrylic acid or its salts

Concentration of these additives is about 0.1 and an anion exchange resin is added together

up to 5 g acrylic acid per liter of culture medium. Especially 5 will.

the other will be a concentration between about 0.5 and

1.5 g of acrylic acid per liter of culture medium are preferred. play explained in more detail.
In a special embodiment of the method

according to the invention, in which only acrylic acid or B e i s ρ i e 1 e 1 to 4 and comparative example 1

whose salts are added, a suitable io

Amount of acrylic acid or its salts 10 minutes 1 g ammonium nitrate, 0.6 g monopotassium phosphate, ^{sterilized at 120 3} C and after the start of the Züch- 4 g dipotassium phosphate, 0.5 g magnesium sulfate, 10 mg ment, for example 1 to 48 hours after the start Ferrous sulfate, 30 mg calcium chloride, 10 / ug manganese cultivation added. Chloride and 100 mg yeast extract were dissolved in one liter. If only an anionic exchange resin or acrylic tap water was dissolved and the solution was adjusted to a pH acid or its salts and an anionic exchange value of 7.0. 50 ml parts of the resin obtained are added, the cultivation can be carried out in the same way solution were in each case in 500 ml shake flasks. pour. 1.5 ml of n-tridecane was added to each flask as After the cultivation is complete, the culture carbon source is introduced and the solution is made acidic, and the resulting space is sterilized at 120.degree.

acids from the culture liquid using As an anionic exchange resin, which the culture

an organic solvent such as ether or medium was added, a weakly basic

Benzene, extracted. The extracted fatty acids are used as an anion exchange resin. The resin

then with a sodium hydroxide solution or was first converted to the hydroxy form by

extracted from another alkali solution, with a strong 25 times the volume of a l-n-sodium hydroxide

! ken acid, such as hydrochloric acid or sulfuric acid solution, was used and then with the

acidified and then washed with an organic lo- lOfold volume of distilled water

solvent such as ether or benzene extracted. After that it was. It was 5 times the amount of a 5 "igen

the solvent is removed by evaporation, phosphate buffer solution (pH 7.0), which is made of monopotassium

where the above-mentioned three fatty acids consisted of 30 phosphate and disodium phosphate, added,

! will. to convert the resin to the phosphate form. That

! If an anion exchange resin is used alone or in addition, resin was then used with ten times the volume

used together with acrylic acid or its salts. washed amount of distilled water. 10 ml each

the culture medium after the end of the cultivation parts of the resin were separated into a 100 ml

Conversion of the ion exchange resin by filtration into 35 Erlenmeyer flasks and introduced during a

separates. The elimination resin is then intermittently at 100 ° C. for a period of 3 days

a mixture of an aqueous solution of a strong sterilized for 30 minutes each time. The sterilized lo-

Acid, such as hydrochloric acid or sulfuric acid, with a solution were added to the culture medium before

water-miscible organic solvents, such as inoculation, added aseptically.

Methanol or acetone, treated to dissolve the fatty acids, 40 Then, Corynebacterium diioxydans adsorbed on the ion exchange resin, ATCC 21766 20 hours in the above. After En of the organic solu- culture medium distant grown and _o aseptically inoculated the solution obtained in sungsmittels from this liquid by evaporating an amount of 5 "., The fatty acids in a similar manner with time as above was shaking at 30 ⁰ C with <> 8 cycles of an organic solvent such as ether or benzene per minute (amplitude 70 mm) and extracted zol., And after removing the solution after 24 hours, 50 ml of 10 minutes at by means of the three fatty acids in raw state 120C sterilized sodium acrylate to 50 ml of the raw fatty acids can also be added aseptically through the culture medium.
Removal of the fatty acids from the aqueous phase The cultivation was terminated 120 hours after the maintenance, which was terminated after the removal of the organic vapor from the cells. The culture medium, the solvent remains. to which neither the ion exchange resin nor sodium · The crude fatty acids are added with a suitable acrylic. (Example 1) and the alcohol, such as methanol, and esterified by a loading culture medium to the only sodium acrylate known separation process, z. B. had been by distillation (Example 2), were made more concentrated under reduced pressure in the dimethyl ester of 55 sulfuric acid and 3nwri with the straight-chain dicarboxylic acid, a methyl ester of the amount of ether corresponding to the culture medium, omega-hydroxy fatty acid and in a methyl ester extracted. The extracts were combined and separated 3 times with (omega-l) -keto fatty acid. extracted from a 1N sodium hydroxide solution. The procedures for obtaining the above-mentioned trium hydroxide-containing solution were made acidic in the same way as acidity from the culture media are not the subject of the invention with concentrated sulfuric acid. <* nd then extracted with ether. The ether The addition of acrylic acid or its salts and was dehydrated with anhydrous sodium sulfate and an anion exchange resin to the culture medium with then the ether removed by evaporation, the method according to the invention are preferred. The crude acids were obtained. Compared with the case that these compounds were not 65 On the other hand, the culture medium to which only added, the amounts of crude fat - the ion exchange resin was added, and acidic became about 1.5 to 2 times higher in the case that the Culture medium to which both the ion exchange acrylic acid or its salts are added, such as resin and the sodium acrylate have been added

was (Example 4), by suction filtering into the culture liquid and the ion exchange resin separately. The resin was placed in a column, and that 40 times its volume of a mixture of equal amounts of 2N hydrochloric acid and methanol were through the Column flowed to elute the fatty acids absorbed on the resin. Was the methanol out Removed from this eluate by evaporation, the eluted fatty acids became insoluble. The extraction with Ether provided the raw acids.

For comparison purposes, the procedure of Example 4 was repeated except that Corynebacterium 7EIC strain (ATCC 13767) used. The results are given under Comparative Example 1.

The crude acids thus obtained were weighed, and the results are shown in Table 1.

10

The acids were methylated with diazomethane, and the content of undecane-l.ll-dicarboxylic acid, 13-hydroxytridecanoic acid and 12-ketotridecanoic acid was measured. The results are given in Table I. The proportion of monocarboxylic acid (tridecanoic acid) was very low.

Dimethylundecane l.ll -dicarboxylate, methyl-13-hydroxytridecanate and methyl 12-ketotridecanate were obtained by gas chromatography, and the

ίο Melting points, mass spectra and the infrared absorption spectra certainly. These esters were saponified to give undecane-l.ll-dicarboxylic acid, 13-hydroxytridecanoic acid and 12-keto-decanoic acid. The melting points, mass spectra and infrared spectra were measured. These measured values corresponded to those of standard products.

Table I.

example 1
without sodium
acrylate,
without ions
replacement resin Example! 2
Sodium acrylate
admitted Example 3
Ions
replacement resin
admitted Example 4
Sodium acrylate
plus ions
replacement resin
admitted Comparative example 1
Sodium acrylate
admitted plus
Ion exchange resin Amount of educated pigs
acid
(g / l culture medium) 3.32 5.05 9.60 16.20 2.43 Salary, determined by
Gas chromatography
Undecane-ljl-dicarbon-
acid (g / l culture medium) 0.72 1.01 2.04 3.85 0.73 13-hydroxytridecanoic acid
(g / l culture medium) 1.48 2.08 4.20 7.24 0.24 12-ketotridecanoic acid
(g; l culture medium) 0.44 0.68 1.16 1.80

Examples 5 to 8

3 g of ammonium chloride, 1 g of monopotassium phosphate, 3.5 g of disodium phosphate, 0.5 g of magnesium sulfate, 10 mg of ferrous sulfate, 30 mg of calcium chloride and 100 mg of meat extract were dissolved in one liter of tap water to form a culture medium. The pH of the culture medium was adjusted to 7.5. 100 ml portions were then placed in 500 ml bulk flasks. To each of these flasks, 3 ml of n-octadecane was added and sterilized at 120 ° C for minutes. Then 5 g of peptone, 2.5 g of meat extract, 2.5 g of yeast extract and 1 liter of tap water were mixed to form a culture medium with a pH of 7.0. Corynebacterium dioxydans ATCC 21766 was grown for 10 hours. The obtained solution with the cells was inoculated into the flasks at an inoculation amount of 2 ° each. inoculated.

A strongly basic anion exchange resin was prepared in the same manner as described in Example 3, sterilized and S ml of the resin were given in 3 portions after 24th, 48 or 72 hours to 100 ml of culture medium each time. Potassium acrylate. in the same Sterilized like sodium acrylic in Example 2.

was used in an amount of 150 mg per 100 ml of culture

medium in 50 mg parts simultaneously with the addition

of the anion exchange resin added. All in all

was cultivated for 150 hours at 25 ° C. with shaking at 98 changes per minute (amplitude 70 mm).

After the completion of the cultivation, it became the same

The treatment as described in Examples 1 to 4 was carried out and the crude acids obtained. These acids were weighed. The results * obtained are shown in Table U. The acids were methylated with diazomethane, and then the content of hexadecane-l-l-dicarboxylic acid, 18-hydroxystearic acid and 17-ketostearic acid was determined. The results are given in Table H. The content of monocarboxylic acid (stearic acid) was very low, dimethylhexadecane-1,16-dicarboxylate. Methyl-18-hy

Droxystearate and methyl-17-ketostearate were durd Gas chromatography separated and recovered. Dw melting points. Mass spectra and infrared spectra were measured. These esters were formed with the formation of hexadecane-1,16-dicarboxylic acid. 18-hydroxystea

ric acid and 17-ketostearic acid saponified, and άν melting points. Mass spectra and infrared spectra of these acids were determined. These values correspond to those of synthesized standard products

, Table II

Example p
without potassium acrylate,
without ions
replacement resin Example 6
Potassium acrylate
admitted Example 7
Ion exchange resin
admitted Examples 8
Potassium acrylate
plus ions
replacement resin
. admitted Amount of crude acids formed
(g / l culture medium)
Content determined by gas
chromatography
Hexadecane-1,16-dicarboxylic acid.
(g / l culture medium)
18-hydroxystearic acid
(g / l culture medium)
17-ketostearic acid
(g / l culture medium) 3.04
0.66
1.38
0.38 4.56
1.02
2.10
0.54 9.46
2.10
4.40
0.98 15.72
3.14
7.10
1.80

Examples 9 to 12

0.5 g urea, 1 g monopotassium phosphate, 3 g disodium phosphate, 0.5 g magnesium sulphate, 10 mg iron (1 IJ sulphate and 100 mg meat extract were dissolved in one liter of tap water and the pH of the solution was adjusted to 6.5 Then 50 ml portions were each placed in 500 ml shake flasks and 1.5 ml n-pentadecane were added to each of these flasks as a carbon source. This was followed by 20 minutes of ^{sterilization at 120 °} C. A spoon of Corynebacterium hydrocarbooxydans nov. sp.ATCC 21767, which had previously been cultivated for 24 hours in a slant culture (pH = 7.0) consisting of 10 g peptone, 5 g meat extract, 5 g yeast extract, 20 g agar and 1 liter tap water, inoculated into the flasks .

As the ion exchange resin, a strongly basic anion exchange resin which had been converted into the hydroxyl form and sterilized in the manner described in Example 3 was used. The addition to the culture medium was carried out in such a way that after 12 hours after the inoculation of the cells, 10 ml of the ion exchange resin was added to each shake flask. Acrylic acid was sterilized for 10 minutes at 120 ^° C. and added to the culture medium in an amount of 25 mg per 50 ml of culture medium 18 hours after the cells had been inoculated. A shaking culture was carried out for 120 hours under shaking conditions of 120 changes per minute

as 30 ⁰ C carried out.

After the completion of the cultivation, the products were prepared in the same manner as in Examples 1 to 4 described, treated. The obtained crude acids were weighed and the results are in Table 111 reproduced.

These products were methylated with diazomethane and the content was determined by gas chromatography of tridecane-1.n-dicarboxylic acid, 15-hydroxypentadecanoic acid and 14-ketopentadecanoic acid measured. The results are shown in Table III. The proportion of monocarboxylic acid (pentadecanoic acid) was very low. Dimethyltridecane-l-.n-dicarboxylate, Methyl 15-hydroxypentadecanate and methyl 14-ketopentadecanate were obtained by gas chromatography and the melting points, mass spectra and Infrared spectra measured. Furthermore, these products were formed with the formation of tridecane-l, 13-dicarboxylic acid, 15-hydroxypentadecanoic acid and 14-ketopentadecanoic acid saponified. The melting points, mass spectra and infrared spectra of these acids were measured The values corresponded to those of synthesized standard products.

Table HI Example 9

without acrylic acid, without ions »

Example 10

Acrylic acid admitted

Example 11

Ion exchange resin

Example 12 Acrylic acid plus

Ion exchange resin

Amount of crude acids formed j
(g / l culture medium) j

Content, determined by gas I.

Tridecane-1,13-dicarboxylic acid
(g / l culture medium)

15-hydroxypentadecanoic acid
(g / l culture medium)

14-KetopentadecaasäuTe
(g / I culture medium)

2.04

1.04
0.04
0.32

3.15

1.42
0.08
0.48

6.20

3.00
0.12
1.05

10.34

5.40 0.16 1.48

Examples 13 and 14 14

2 g of ammonium acetate, 0.6 g of monopotassium phosphate, 4 g of disodium phosphate, 0.5 g of magnesium phosphate, 10 mg of Eixen (t]) sulfate, 30 mg of calcium chloride, 10 μ% of manganese chloride, 100 mg of yeast extract and 1 liter of tap water were used to form a Culture medium mixed. In each case 15 liters of the nutrient medium were placed in two 30 liter fermentation vessels. After adjusting the pH to 7.0, 450 ml of a mixture of 70% n-pentadecane and around 30% n-hexadecane were added. It was then sterilized with water at 120 ^{° C. for 20 minutes.} Then 5 percent by volume of a cell fluid obtained by preculturing Corynebacterium dioxydans nov. sp. ATCC 21766 was aseptically inoculated into the two 30 liter fermentation vessels.

3 liters of a weakly basic anion exchange resin were prepared as described in the same manner in Example 3, treated, and added after 20 minutes of sterilization at 120 ¹ C to a fermentation vessel. No anion exchange resin was added to the other fermentation vessel. There was a breeding, was stirred at the through air, carried out at 3O ⁰ C, wherein the air flow rate was 10 liters per minute and the stirring speed was 250 rpm. 24 hours after the inoculation, 15 g of sodium acrylate, which had been sterilized at 120 ° C. for 10 minutes, was added to the fermentation vessel in which the ion exchange resin was located. The cultivation was continued for another 96 hours. The fermentation vessel containing no ion exchange resin and sodium acrylate was subjected to cultivation for 120 hours.

After the completion of the cultivation, the crude acids were weighed. The acids were melhylated and the amounts of tridecane-1-D-dicarboxylic acid, tetradecane -1,14 - diwboxylic acid, 15 - hydroxypentadecanoic acid, 16-hydroxypalmitic acid, 14-ketopentadecanoic acid and 15-ketopalmitic acid were measured in a similar manner. The results are in Table IV reproduced. The proportions of monocarboxylic acids, pentadecanoic acid and palmitic acid were very low.

Dimethyltridecane-1,13-dicarboxylate, dimethyltetradecane -1,14 - dicarboxylate, 15 - hydroxypentadecanate and methyl 16-hydroxypalmitate, 14-methylketopentadecanate and methyl 15-ketopalmitate were by Gas chromatography obtained and the melting points, mass spectra and infrared spectra measured. These esters were saponified to give tridecane-1,13-dicarboxylic acid, Tetradecane-l, 14-dicarboxylic acid, 15 - hydroxypentadecanoic acid, 16 - hydroxypalmitic acid, 14-ketopentadecanoic acid and 15-ketopentadecanoic acid to obtain. The melting points, mass spectra and infrared spectra were measured. These values corresponded to those of standard synthesized products.

Table IV

Amount of educated

ao raw acids
(g / 15 1 culture medium)

Content, determined by gas chromatography

»5 graphic

Tridecane-1,13-dicarboxylic acid (g / 15 1 culture medium)

Tetradecane-1,14-dicarboxylic acid (g / 15 l culture medium)

15-hydroxypentadecanoic acid

(g / 151 culture medium)

16-hydroxypalmitic acid

♦ o (g / 151 culture medium)

14-ketopentadecanoic acid
(g / 15 1 culture

medium)

15-ketopalmitic acid (g / 15 1 culture medium)

Example without sodium acrylate,

ol'ine ion exchange resin

45.1

6.8

2.9

14.1

6.1

4.3 1.8

Example nairium acrylate

plus ion exchange resin

admitted

231.4

33.8 14.5 70.4 30.5

21.5 8.5

Claims (1)

fatty acid, straight-chain dicarboxylic acid and (omega-1) claims: ketocarboxylic acid on an industrial scale from n-paraffins using microorganisms 1. Microbiological process for simultaneous delivery. gen production of a straight-chain dicarboxylic acid, 5 Accordingly, the object of the invention is to create an omega-hydroxy fatty acid and a (ome- drive) in which at the same time a straight D-keto fatty acid, each of these acids being the same as the chain dicarboxylic acid, an omega-hydroxy fatty acid Number of carbon atoms has like that and an (omega-1) -ketofatty acid in high yield n-paraffin substrate in the culture medium, characterized by η-paraffins using an n-paraffin, characterized in that one Corynebacte- io assimilating strain that has the ability to form these rium dioxydans nov.sp.ATCC 21 766 or Cory acids at the same time.
nebacterium hydrocarbooxydans nov. sp. ATCC There were two new η-paraffin assimilating 21 767 in a culture medium, which a stick strains of the genus Corynebacterium, which possess the fiber source, mineral salts and an η-paraffin with ability, at the same time straight-chain dicarbons at least 10 carbon atoms, among »5 acids, breeds omega-hydroxy fatty acids and (omega-1) aerobic conditions. To form ketocarboxylic acid in large quantities, 2, method according to claim 1, characterized thereby. records that the culture medium also contains a These 2 strains, Corynebacterium dioxydans nov. Additive from the group of acrylic acid, the sp. ATCC217 & 5 and Corynebacterium hydrocarbo salts and anion exchange resins. 20 oxydans ATCC 21767 differ from the ones before known η-paraffin assimilating strains. You have a distinct ability in certain ways to have an oxidizing effect (diterminal oxidation), while they assimilate n-paraffin as a carbon source, 25 and give and accumulate large amounts of The invention relates to a microbiological straight-chain dicarboxylic acids, omega-hydroxy fatty acids for the simultaneous production of a straight chain acids and (omega-1) -Ketocarbonsäuren that the gen dicarboxylic acid, an omega-hydroxy fatty acid with the same number of carbon atoms as the substrate in the and an (omega-l) keto fatty acid, each of which have culture medium. In particular, these have Acids the same number of carbon atoms possesses 30 2 strains the unique ability to be omega-hydroxy like the n-paraffin substrate in the culture medium. to enrich fatty acids in large quantities. This is It is known that fatty acids from η-paraffins surprisingly, since they have so far been very unstable Microorganisms are formed, e.g. B. have been viewed by cross-intermediates, and it Yeasts of the genus Pichia (Agr. Biol. Chem. 29, 1009, it was difficult to obtain them in substantial quantities 1965), by the strain 7EIC (ATCC 13 767) of a 35 brazen. unknown species belonging to the genus Corynebacterium The present invention thus consists in one heard that differ from the two microorganisms - microbiological processes for simultaneous Her-strains, in the method according to the invention position of a straight-chain dicarboxylic acid, a can be used (see below), with regard to size and omega-hydroxy fatty acid and an (omega-l) -keto form and such properties as the fermentability 40 fatty acid, each of these acids having the same number differ from carbohydrates (J. Bacteriol, 85, has carbon atoms like the n-paraffin substrate 859, 1963), by bacteria of the genus Pseudomo- in the culture medium, characterized in that one nas (Nature, 198, 289, 1963) by yeasts of the genus Corynebacterium dioxydans nov. sp. ATCC 21766 Candida (J. Gen. Appl. Microbiol, 12, 119, 1966) and or Corynebacterium hydrocarbooxydans nov. sp. by molds of the genus Botrytis (J. of 45 ATCC 21767 in a culture medium containing a stick-Japan. Society of Agricultural Chemistry, 40, 364, stoffquelle, mineral salts and an η-paraffin with we-1966). At least 10 carbon atoms under aerobic. In these publications it is only described conditions breeding. that omega-hydroxy fatty acids, dicarboxylic acids or furthermore it was found that when adding Ketocarboxylic Acids as Metabolic Intermediates 50 agreed additives that are known to be while the assimilation of η-paraffins by them alone can be applied to others Microorganisms can be detected. Products using microorganisms In J. Gen. Appl. Microbiol, 12, 119 (1966) can be obtained from other genera to which, in the case of the prescription, that in the cultivation of Candida rugosa drive according to the invention used culture JF 101 in a medium, the 2.5 ml η-paraffin per 55 medium, the above 3 acids in large Containing 80 ml of an inorganic salt solution, 16.7 mg quantities are formed and these acids are similar Dicarboxylic acid per liter of culture liquid can be formed early with increased yield, became. It was, however, the general view that it was also found that the common The turn of these particular additives into a particularly high amount of fatty acid, formed by microorganisms from η-paraffin, is very small. In particular, it leads 60 significant improvements
difficult to enrich an omega-hydroxy fatty acid, For example, it was known that dicarboxylic acids, because it is an unstable metabolic intermediate. derived from η-hexane and n-heptane, by resting It was not previously known that an omega-Hy cell from Pseudomonas could be produced using hydroxy fatty acid, a straight-chain dicarboxylic acid and acrylic acid are obtained (Biochim. Biophys. Acta, one (omega-1 ^ ketocarboxylic acid simultaneously in large 65 84, 195, 1964). It is also known that p-ToluoI amounts are formed. carboxylic acid from p-xylene by Actynomyces under η-paraffins are easily available at low cost, using an ion exchange resin is formed and it is therefore desirable to use omega-hydroxy (Biotechnol. Bioeng. 10, 689, 1968).